Determination of Xipamide metabolite in human urine by high-performance liquid chromatography/diode-array detection, high-performance liquid chromatography/electrospray ionization mass spectrometry and gas chromatography/mass spectrometry

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<ul><li><p>RAPID COMMUNICATIONS IN MASS SPECTROMETRY</p><p>Rapid Commun. Mass Spectrom. 2004; 18: 25052512</p><p>Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1636</p><p>To the Editor-in-Chief</p><p>Sir,</p><p>Determination of Xipamide metabo-</p><p>lite in human urine by high-perfor-</p><p>mance liquid chromatography/diode-</p><p>array detection, high-performance</p><p>liquid chromatography/electrospray</p><p>ionization mass spectrometry and gas</p><p>chromatography/mass spectrometry</p><p>Diuretic agents are drugs that increase</p><p>renal excretion of water and solutes</p><p>(mainly sodium salts). The major pur-</p><p>poses of diuretic therapy are to</p><p>decrease fluid volume of the body and</p><p>to adjust the water and electrolyte bal-</p><p>ance. Diuretics are drugs widely used</p><p>in clinical practice mainly in the treat-</p><p>ment of hypertension and in different</p><p>kinds of edema.13</p><p>Recently, diuretics have been abused</p><p>in sports with weight classes, such as</p><p>weightlifting, wrestling and boxing.</p><p>Athletes try to reduce their body</p><p>weight in order to qualify for lower</p><p>weight classes. It is also reported that</p><p>athletes use diuretics to avoid detection</p><p>of doping agents by reducing their</p><p>urine concentration.4 As part of their</p><p>efforts to fight drug abuse in sports, the</p><p>Medical Commission of the Interna-</p><p>tional Olympic Committee has banned</p><p>diuretics since the 1988 Seoul Olympic</p><p>Games.</p><p>Diuretics may be classified accord-</p><p>ing to their chemical structure, mecha-</p><p>nism, primary site of action in the</p><p>nephron and their diuretic potency.5</p><p>These diuretics exert their effects by</p><p>inhibiting tubular sodium and water</p><p>reabsorption by epithelial cells lining</p><p>the renal tubule system. Certain diure-</p><p>tics (such as carbonic anhydrase inhi-</p><p>bitors, loop diuretics, thiazide-like</p><p>diuretics and potassium-sparing diure-</p><p>tics) suppress sodium and water reab-</p><p>sorption by inhibiting the function of</p><p>specific proteins that are responsible</p><p>Copyright # 2004 John Wiley &amp; Sons, Ltd.</p><p>RCM</p><p>Letter to the Editor</p><p>Table 1. HPLC/DAD operating conditions</p><p>HPLC conditionsFlow rate: 1.0 mL/minMobile phase solvent A: Phosphate buffer (pH 6.8)</p><p>solvent B: AcetonitrileGradient timetable:Time (min) 0 10 15 18Solvent A (%) 96 70 55 50Solvent B (%) 4 30 45 50Detection wavelength: 232 nmInjection volume: 5mLColumn temperature: 408CColumn: Hypersil-ODS C18 column (4.6 mmi.d. 100 mm, particle size 5mm)</p><p>Table 2. HPLC/ESI-MS operating conditions</p><p>HPLC conditions</p><p>Flow Rate: 0.2 mL/minMobile Phase solvent A: Acetonitrile</p><p>solvent B: 20 mM Ammonium formate (pH 4)Gradient timetable:Time (min) 0 5 18Solvent A (%) 20 50 50Solvent B (%) 80 50 50Injection Volume: 5mLColumn Temperature: 408CColumn: Capcell Pak C18 column (MG type, 2.0 mmi.d. 150 mmparticle size 5mm)MS conditionsIonization: ESI (electrospray ionization)Mode: Positive ionMass range: m/z 50420Nebulizing Gas Pressure: 30 psi (N2)Drying Gas Temp: 3508CDrying Gas Flow: 8.0 L/minCapillary Exit Voltage: 60.0 eVMS/MS conditions</p><p>Collision gas: He (70 psi)Collision energy: 0.8 eVIsolation width of parent ion: 1.5 Da</p><p>Table 3. GC/MS operating conditions</p><p>GC conditionsCarrier gas: He at 1.0 mL/minOven temp. program:</p><p>Initial temp.(8C)</p><p>Initialtime (min)</p><p>Rate(8C/min)</p><p>Final temp.(8C)</p><p>Final time(min)</p><p>180 1 15 300 5</p><p>Injection volume: 2mLSplit mode: Split (1:10)Injection port temp.: 2808CTransfer line temp.: 2808CColumn: Ultra-2 (cross-linked 5 %phenylmethylsiloxane, 0.2 mmi.d 17 m length 0.33mmfilm thickness)MS conditions</p><p>Ionization: EISource temp.: 2008CAcquisition mode: scanMass range: m/z 50600</p></li><li><p>for (or participate in) the transportation</p><p>of electrolytes across the epithelial</p><p>membrane; osmotic diuretics inhibit</p><p>water and sodium and water reabsorp-</p><p>tion by increasing intratubular osmotic</p><p>pressure. Different types of diuretics</p><p>may inhibit different transporters in dif-</p><p>ferent segments of the tubular system.6,7</p><p>Xipamide (4-chloro-5-sulfamoyl-20,60-salicyloxylidide) is a diuretic drug</p><p>used for the treatment of high blood</p><p>pressure and a edema of cardiac,</p><p>hepatic or renal origin. It is a non-</p><p>thiazide diuretic with a greater anti-</p><p>hypertensive effect and may cause a</p><p>lower potassium loss relative to</p><p>sodium excretion than these drugs.</p><p>Xipamide offers a suitable alternative</p><p>to other diuretics in the treatment of</p><p>patients with mild to moderate hyper-</p><p>tension and of patients with edema due</p><p>to a variety of causes.8</p><p>Metabolite monitoring is important</p><p>for the determination of any medica-</p><p>tion by drugs. Several chromatogra-</p><p>phic methods have been reported for</p><p>the separation, detection, and quanti-</p><p>tative measurement of individual</p><p>diuretic agents in biological fluids. Pub-</p><p>lished methods include those based</p><p>on thin-layer chromatography (TLC),9</p><p>gas-liquid chromatography (GLC),10</p><p>gas chromatography/mass spectro-</p><p>metry (GC/MS),10,11 high-performance</p><p>liquid chromatography (HPLC),1216</p><p>and high-performance liquid chroma-</p><p>tography/mass spectrometry (HPLC/</p><p>MS).17 Generally, metabolites in urine</p><p>are polar, so it is not easy to use GC/MS</p><p>without derivatization. More recently,</p><p>HPLC with detection by tandem mass</p><p>spectrometry (HPLC/MS/MS) has</p><p>been used for trace level bioanalysis.18</p><p>This technique allows highly sensitive</p><p>determinations without the need for</p><p>derivatization as required for GC/MS.</p><p>It is also possible to devise methods</p><p>specific for metabolites and structurally</p><p>similar compounds.</p><p>This paper describes the HPLC/</p><p>diode-array detection (DAD) screening</p><p>procedure and HPLC/MS confirma-</p><p>tion method of Xipamide in human</p><p>urine for doping control tests. The</p><p>metabolite of Xipamide was also stu-</p><p>died by GC/MS after methylation for</p><p>characterization.</p><p>Xipamide tablets were purchased</p><p>from Bukwang Pharm. Co., Ltd. (Seoul,</p><p>Korea). Anhydrous sodium sulfate was</p><p>Scheme 1. Structure of Xipamide.</p><p>Figure 1. HPLC/DAD chromatograms of Xipamide in (a) spiked urine, (b) blank</p><p>urine, and (c) dosed urine.</p><p>Copyright # 2004 John Wiley &amp; Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 25052512</p><p>2506 Letter to the Editor</p></li><li><p>purchased from Sigma Chemical Co.</p><p>(MO, USA) and HCl was from Merck</p><p>(Darmstadt, Germany). Diethyl ether,</p><p>ethyl acetate and acetone were HPLC</p><p>grade from J. T. Baker (Phillipsburg,</p><p>NJ, USA). Acetonitrile and methanol</p><p>from J. T. Baker and distilled water</p><p>were used after filtering through Milli-</p><p>pore filters (0.5 and 0.45 mm, respec-tively) and sonication for 20 min. The</p><p>internal standard (ethyltheophylline)</p><p>was prepared at the Doping Control</p><p>Center (KIST, Korea)</p><p>A Hewlett-Packard (Palo Alto, CA,</p><p>USA) HP 1100 series liquid chromato-</p><p>graph, coupled with a HP 1100 series</p><p>G1315A diode-array detector, was</p><p>used to screen Xipamide and its meta-</p><p>bolite. The column for HPLC was a</p><p>Hypersil-ODS C18 column (4.6 mm</p><p>i.d. 100 mm, particle size 5mm, Ther-mohypersil). The HPLC/ESI-MS sys-</p><p>tem consisted of an HP 1100 series</p><p>binary pump HPLC system (Agilent,</p><p>Palo Alto, CA, USA) and an LC/MSD</p><p>ion trap equipped with an electrospray</p><p>ionization (ESI) source. It was used for</p><p>confirmation of Xipamide and charac-</p><p>terization of suspected metabolites.</p><p>The column for HPLC/ESI-MS was a</p><p>Capcell Pak C18 column (MG type,</p><p>2.0 mm i.d. 150 mmparticle size5 mm, Shiseido). A Trace GC-Polaris Q(Finnigan Inc., San Jose, CA, USA) was</p><p>used for reconfirmation of the suspec-</p><p>ted metabolite. The column for GC/</p><p>MS was an Ultra-2 capillary column</p><p>(0.2 mm i.d. 17 m length 0.33 mmfilm thickness, Agilent Technologies).</p><p>A Lauda (Lauda-Konigshofen, Ger-</p><p>many) Ecoline RE112 freezer was used</p><p>to freeze the aqueous layer. A Turbo-</p><p>vap1 LV evaporator supplied by</p><p>Zymark Corporation (Hopkinton,</p><p>MA, USA) was used to evaporate the</p><p>extracted organic solvents.</p><p>A healthy male volunteer (age 29,</p><p>weight 70 kg) was dosed with one</p><p>Diurexan tablet (Xipamide, 20 mg/</p><p>tab). Urine samples were collected for</p><p>48 h and subjected to the screening</p><p>procedure; these urine samples were</p><p>kept at 238C.Anhydrous sodium sulfate (1 g) was</p><p>added to the urine (pH 5 adjusted with</p><p>phosphate buffer). After vortexing,</p><p>5 mL distilled diethyl ether were</p><p>added. After shaking for 20 min and</p><p>centrifugation at 2500 rpm for 5 min,</p><p>the solution was frozen in the freezer</p><p>(308C). The organic phase was trans-ferred to another tube and evaporated</p><p>to dryness at 408C under a gentlestream of nitrogen. The residue was</p><p>then reconstituted with 200 mL metha-nol, of which 5 mL were injected on theHPLC/DAD and LC/MS system.</p><p>Then, 150mL of the residue were driedunder a nitrogen gas stream, and 50mLof methyl iodide and 150 mL of acetonewere added and then vortex-mixed.</p><p>The mixture was heated at 808C for 1 hand then cooled to room temperature.</p><p>Finally, 2 mL of this solution wereanalyzed by the GC/MS system.</p><p>Operating conditions for HPLC/</p><p>DAD and HPLC/ESI-MS, used for the</p><p>detection of Xipamide and the char-</p><p>acterization of its metabolite, are</p><p>described in Tables 1 and 2. Mass</p><p>spectrometry (MS) and tandem mass</p><p>spectrometry (MS/MS) analyses were</p><p>performed on a LC/MSD ion trap mass</p><p>spectrometer for confirmation. The</p><p>entire column eluent was directly</p><p>introduced into an ESI interface</p><p>through a 50 cm long PEEK tubing</p><p>(0.13 mm i.d.). Operating conditions</p><p>for GC/MS are described in Table 3.</p><p>The molecular mass of Xipamide is</p><p>354.04 Da; its structure is shown in</p><p>Scheme 1. It is a sulfonamide diuretic</p><p>with a high boiling point and polarity.</p><p>HPLC/DAD chromatograms of stan-</p><p>dard spiked urine, blank urine, and</p><p>the urine sample from the dosed</p><p>volunteer are shown in Fig. 1. We</p><p>found unmetabolized Xipamide and</p><p>one unknown peak, designated Xipa-</p><p>mide-M1, at a shorter retention time</p><p>(6.23 min) than Xipamide (8.91 min), in</p><p>the dosed urine. Figure 2 shows the</p><p>ultraviolet spectra of the unknown</p><p>peak (Xipamide-M1) and of Xipamide</p><p>itself; these spectra are very similar.</p><p>From these results, the metabolite of</p><p>Xipamide was suspected to be a</p><p>compound of similar structure but</p><p>substituted by a polar functional</p><p>group such as hydroxyl to account</p><p>for the reduced retention in reversed-</p><p>phase HPLC.</p><p>[MH] ions were mainly producedin positive ESI mode, and [MNa]and/or [MK] ions were also pro-duced by impurities in the solvent.</p><p>Figure 3 shows the total ion chromato-</p><p>gram (TIC), extracted ion chromato-</p><p>gram (EIC), mass spectrum, and the</p><p>MS/MS spectrum of the [MH]</p><p>Figure 2. UV spectra of (a) Xipamide and (b) Xipamide-M1 from dosed urine</p><p>obtained by HPLC/DAD.</p><p>Letter to the Editor 2507</p><p>Copyright # 2004 John Wiley &amp; Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 25052512</p></li><li><p>Figure 3. (a) Total ion chromatogram, (b) extracted ion chromatogram for [MH] and(c) MS and (d) MS/MS spectra of Xipamide standard, obtained by HPLC/ESI-MS.</p><p>2508 Letter to the Editor</p><p>Copyright # 2004 John Wiley &amp; Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 25052512</p></li><li><p>Figure 4. (a) Total ion chromatogram, (b) extracted ion chromatogram for [MH] and(c) MS and (d) MS/MS spectra of Xipamide-M1, obtained by HPLC/ESI-MS from dosed</p><p>urine.</p><p>Letter to the Editor 2509</p><p>Copyright # 2004 John Wiley &amp; Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 25052512</p></li><li><p>ion, of the Xipamide standard. The</p><p>spectrum of the standard (Fig. 3(b))</p><p>includesm/z 355 ([MH]) andm/z 377([MNa]), and shows the isotopepattern characteristic of the Cl atom</p><p>(m/z 357). Figure 3(d) shows the</p><p>MS/MS spectrum of m/z 355 from</p><p>Xipamide as the precursor ion, and</p><p>major fragment ions atm/z 338, 274 and</p><p>290 were detected in addition to less</p><p>abundant fragments at m/z 210 and</p><p>122.</p><p>If the metabolism of Xipamide leads</p><p>to hydroxylation (insertion of an O-</p><p>atom), the molecular mass of Xipa-</p><p>mide-M1 is expected to be 370.04 Da.</p><p>We detected Xipamide-M1 in urine</p><p>from the dosed volunteer by HPLC/</p><p>ESI-MS. Figure 4 shows the TIC, EIC for</p><p>the [MH] ion at m/z 371, the massspectrum and MS/MS spectrum. The</p><p>mass spectrum of Xipamide-M1 shows</p><p>the expected [MH] ion at m/z 371and the sodium adduct ion [MNa] atm/z 393 (Fig. 4(b)). We could not</p><p>determine from this information where</p><p>the additional hydroxyl group was</p><p>Figure 5. GC/MS chromatograms of methylated Xipamide in (a) spiked urine, (b) blank</p><p>urine, and (c) dosed urine.</p><p>2510 Letter to the Editor</p><p>Copyright # 2004 John Wiley &amp; Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 25052512</p></li><li><p>substituted, so MS/MS was applied to</p><p>obtain information about the position</p><p>of the hydroxyl group. Figure 4(c)</p><p>shows the MS/MS spectrum of m/z</p><p>371 from Xipamide-M1. The main frag-</p><p>ment ions were observed at m/z 354</p><p>([MHH2O]), 290 ([MHSO2NH2]</p><p>),275, and 137. Xipamide and Xipamide-</p><p>M1 are much alike in fragmentation</p><p>pattern. These results suggested that</p><p>Xipamide-M1 contained one additional</p><p>hydroxyl group, but did not provide</p><p>any information concerning the posi-</p><p>tion of the hydroxyl group.</p><p>We also used GC/MS for character-</p><p>ization of the metabolite of Xipamide.</p><p>Figure 5 shows the GC/MS chro-</p><p>matograms obtained of methylated</p><p>Xipamide in spiked urine, balnk urine</p><p>and dosed urine. The derivatization</p><p>(methylation) method was applied to</p><p>the dosed human urine, and both</p><p>Xipamide and its metabolite were</p><p>detected. Figure 6 shows the EIC and</p><p>mass spectrum of the methylated</p><p>Xipamide standard obtained by GC/</p><p>MS.</p><p>The mass spectrum (Fig. 6(b)) of</p><p>methylated Xipamide shows the mole-</p><p>cular ion [M] at m/z 410 and the basepeak at m/z 289 ([MCH3, -C8H10]</p><p>).Thus the [M] (m/z 410) ion showsthat four methyl groups were substi-</p><p>tuted in the Xipamide standard (4Me-</p><p>Xipamide). The main fragment ions</p><p>were observed at m/z 303 ([M</p><p>SO2N(CH3)2]), 276 ([MC9H12N]</p><p>)and 233 ([MC9H12NN(CH3)]</p><p>).Figure 7 shows the EIC of the [M]</p><p>ion atm/z 440 and the mass spectrum of</p><p>Xipamide-M1, obtained by GC/MS</p><p>from dosed human urine. The m/z 440</p><p>ion, which was 30 Da more than the</p><p>molecular ion of 4Me-Xipamide, indi-</p><p>cates both hydroxylation and methyla-</p><p>tion of Xipamide. Thus Xipamide-M1</p><p>has one additional hydroxyl group</p><p>compared with Xipamide, even though</p><p>its position could not be determined.</p><p>However, we could expect the addi-</p><p>tional hydroxyl group to appear in the</p><p>dimethylbenzene group, based on</p><p>observation of m/z 289 in the mass</p><p>spectrum of the Xipamide standard</p><p>(Fig. 6(b)) and of dosed urine (Fig. 7(b)).</p><p>In conclusion, we have evaluated a</p><p>screening method and a confirmation</p><p>method for Xipamide in human</p><p>urine. When we compared the chro-</p><p>matograms of the blank urine, spiked</p><p>urine and dosed urine by HPLC/DAD,</p><p>we found an unknown metabolite that</p><p>was shown to be 16 Da heavier than</p><p>Xipamide by HPLC/MS and GC/MS.</p><p>These results suggested that one</p><p>hydroxyl group substitutes to Xipa-</p><p>mide as the metabolite. Even though</p><p>the exact position of hydroxylation</p><p>could not be determined, we can apply</p><p>these observations to confirm Xipa-</p><p>mide in human urine, and these results</p><p>will assist doping control.</p><p>Figure 6. (a) Extracted ion chromatogram for [MH] and (b) MS spectrum ofmethylated Xipamide standard obtained in dosed urine by GC/MS.</p><p>Letter to the Editor 2511</p><p>Copyright # 2004 John Wiley &amp; Sons, Ltd. Rapid Commun. Mass Spectrom. 2004; 18: 25052512</p></li><li><p>AcknowledgementThis research was supported in 2004 by theKyung Hee University Research Fund(KHU-20031102).</p><p>Yunje Kim1*, Kyungjin Han1,2</p><p>and Ki-Jung Paeng2</p><p>1Doping Control Center,Korea Institute of Science and</p><p>Technology, P.O. Box 131,Cheongryang, Seoul, Korea</p><p>2Department of Chemistry,College of Science,</p><p>Yonsei...</p></li></ul>

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